Biomineralization is a complex process through which mineral crystals (apatite in the case of vertebrate bone and dentin) are deposited in an oriented fashion on an organic matrix. The overall goal of this project continues to b elucidation of the mechanism(s) by which vertebrate apatite crystal deposition and growth are regulated. Detailed knowledge about the mechanism(s) will suggest interventions that might be used to prevent craniofacial and musculoskeletal abnormalities associated with defective mineralization. Two hypotheses will be tested. (I)The functions of phosphorylated proteins/glycoproteins in the mineralizing matrix are determined by the nature and extent of their post-translational modifications, the conformation conferred by these modifications, and the modified proteins' interactions with calcium ions and mineral crystals. (II) Because these matrix proteins interact with one another, as well as with mineral crystals, they may affect biologic calcification in combination in ways that are distinct from their effects when acting alone. The specific aims are thus (1) to continue the evaluation of the mechanism(s) of action of bone and dentin phosphoproteins, their under- and over-phosphorylated forms, and their under- and normally-glycosylated forms, and (2) to validate a "combinatorial chemistry" approach for evaluating effects of combinations of proteins on in vitro mineralization. Aim 1 will be achieved by analyzing the in vitro effects on apatite formation and growth of two purified native bone and tooth matrix proteins (osteopontin [OPN] and dentin matrix protein-1 [DMP-1]). Experiments will use the dynamic gelatin gel system, transmission electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy (FTIR). Comparisons will be made to recombinant proteins with or without post-translational modification produced either chemically or in culture, or to native proteins that are de-phosphorylated and/or de-glycosylated. Mechanisms will be explored by monitoring the protein conformational changes (circular dichroism and FTIR) occurring in the presence of Ca+2 or apatite crystals. Verification of predicted in vivo actions will be based on mineral and matrix alterations in bones and teeth of animals lacking these proteins (e.g. OPN and BSP knockouts) or in which these proteins are altered or over-expressed (transgenics) using FTIR microspectroscopy and IR imaging. Aim 2 will consider the interactions of the phosphorylated matrix proteins with other physiologically relevant ECM proteins using a newly developed rapid throughput system (e.g. OPN and osteocalcin; OPN and collagen; OPN and BSP; OPN and fibronectin).